The present invention is related to the biopharmaceutical area, in particular to the production of the human growth hormone (HGH) that has been successfully used in treating different health problems. Specifically speaking, it is related to the use of recombinant DNA technology for the production of recombinant human growth hormone (rHGH) in methylotrophic yeast transformed with the cDNA sequence of mature HGH and the S. Cerevisiae alpha-mating factor pre-pro sequence which, under the regulation of a methanol inducible promoter region of a methylotrophic yeast gene and grown under certain conditions, permit the expression and secretion of rHGH towards the culture medium. The invention further relates to said methylotrophic yeast, the DNA fragments and the vectors used in said transformation.
The problems in which HGH is currently used as a part of therapy are: the treatment of pituitary dwarfism (caused by malfunctioning of the hypophysis); removal of fatty acids and hence the treatment of obesity; improvement of vascularization; promotion of muscle mass gain in elderly people; the treatment of ulcers and bone fractures, and the acceleration of the regeneration of damaged tissues.
Human growth hormone (HGH) or somatotropin is a globular polypeptide of 191 amino acids without sugar residues, with a molecular weight of approximately 22 kDa. Taking into account the three dimensional model of the pig growth hormone, HGH is considered to be made up of 4 antiparallel alpha helices. The molecule has two disulfide bonds that connect helix 4 with the carboxyl terminal end of the protein and helix 1 with helix 4 (Devos et al., 1992).
The production of HGH in the hypophysis is episodical and is governed by the action of two hypothalamic hormones, the growth hormone releasing factor and somatostatin. While the former stimulates its synthesis, the later inhibits its release. The hormone acts directly on the target tissue cells, or by means of intermediaries of hormonal type called somatomedins (Daughaday, 1985). The catabolic effects of HGH include: increase in lipolysis, oxidation of fatty acids and decreased transportation of glucose, with a consequent increase in the concentration of glucose in the blood (Daughaday, 1985). The anabolic effects include: increase in the transportation of amino acids with a decrease in their catabolism and stimulation of the synthesis of DNA, RNA and proteins in the liver (Campbell, 1988). In general terms, the net effect of HGH and of somatomedins is considered to be anabolic (Welsh, 1985). Among the important functions of HGH is its role in post-natal growth.
The importance of HGH and its application in the biopharmaceutical area is reflected in the uses it has had: it has been used successfully in the treatment of pituitary dwarfism; it removes fatty acids and is therefore used in the treatment of obesity; it improves vascularization and promotes muscle mass gain in the elderly; it is used in the treatment of ulcers and bone fractures and accelerate the regeneration of damaged tissues (Watson, 1991).
Recombinant HGH was synthesised for the time in Escherichia coli (E. coli) (2.4 mg/L of culture medium) by Goeddel and coworkers in 1979 under the control of the lac promoter (Goeddel et al., 1979). Secretion of the hormone into the periplasm of the bacteria was achieved by Gray and coworkers in 1985 whose strategy consisted of using the cDNA of HGH fused to the region encoding the signal peptide of the alkaline phosphatase gene to E. coli, making it possible to produce 0.28 mg/L of the hormone, 82% of which was directed to the periplasmic space (Gray, et al., 1985). Becker and Hsiung in 1986 achieved a concentration of 15 mg/L of HGH in the periplasmic space thanks to the outer membrane protein (OMP) peptide signal of E. coli. In 1987, Chang et al. fused the cDNA of HGH to the region encoding the enterotoxin II signal peptide of E. coli using the alkaline phosphatase promoter of this same bacterium produced 15.4 mg/L of HGH, 90% of which was exported to the periplasm. Kato et al., in 1987, reported the synthesis of HGH and its secretion into the culture medium, using a plasmid carrying both the HGH hybrid and the kill genes. The kill gene expresses a protein that permeabilized the outer membrane. The HGH concentration reached was 20.5 mg/L of which 11.2 mg/L were secreted into the medium and 8.6 mg/L remained in the periplasm. Hsiung et al., in 1989, used the bacteriocin releasing protein to allow recombinant HGH, which had been secreted into the periplasm thanks to the presence of the OMP signal peptide, to be released into the culture medium with a concentration of up to 69.6 mg/L. In addition to the E. coli, recombinant HGH has also been produced in B. subtilis, S. cerevisiae and mammalian cells. However, recombinant HGH synthesis in mammalian cells has given very low yields (in the order of xcexcg/L) (Ortiz-Lxc3x3pez, 1992; and Pavlakis et al., 1991).
Yeast offer advantages over bacteria in heterologous protein production because, although they are unicellular organisms easy to manipulate and grow quickly, their cellular organization is eukaryotic, making it possible to perform expression and maturation processes characteristic of animal and plant cells. Moreover they can secrete recombinant proteins into the culture medium, being recombinant product levels higher there than in the cytoplasm. Evenmore, the secreted products are obtained with a high degree of purity (since few endogenous proteins are secreted) and therefore the purification steps are reduced. Finally, they offer a suitable environment for the adequate folding of proteins, especially of those that contain disulfide bonds.
Pichia pastoris is a yeast that can metabolize methanol as the sole source of carbon and energy (methylotrophic) and is currently used for the production of recombinant proteins since as a production system it is simpler, cheaper and more productive than other higher eukaryotic systems. Being a yeast, it shares the advantages of easy genetic and biochemical manipulation of S. cerevisiae but surpasses its heterologous protein production levels (10 to 100 times greater) (Cregg et al., 1993; and Romanos, 1995). Furthermore, expression systems have been developed in P. pastoris that overcome the problems associated with S. cerevisiae, such as how to increase the number of copies of the heterologous gene and genetic stability on scaling up. This system is characterized by the use of a promoter that controls the expression of the heterologous gene. This promoter is derived from a yeast gene that can be induced with methanol and that is subject to precise regulation, often leading to high levels of heterologous gene expression.
P. pastoris has been used in the production of several heterologous proteins, for example the hepatitis B surface antigen (Cregg, et al., 1987), Iysozyme and invertase (Digan, 1988; Tschopp, 1987), tumour necrosis factor (Sreekrishna, 1989), among others. However, efforts to produce other products from heterologous genes in Pichia pastoris, especially via secretion, have given variable results, and there are examples of low yields, proteolysis of the secreted protein, inefficient secretion and in some cases the protein product has not been detected (Romanos, 1995). With the studies done to date in this respect, it is difficult to predict whether one protein in particular will be synthesized and/or secreted by a methylotrophic yeast or if this yeast will tolerate the presence of the produced heterologous protein; likewise, it is also difficult to predict if these processes will be efficient.
Considering 1) that the production levels of a recombinant protein in cultures at flask level do not accurately reflect the production levels observed at the fermenter level; 2) that according to the literature on P. pastoris, these levels rise considerably when P. pastoris is grown on a large scale; 3) the availability of technology for fermentation at high cell densities for P. pastoris; and 4) the high production levels and efficient secretion of recombinant HGH that surprisingly characterizes the expression system described for the first time in this invention, the biotechnological development described here offers important technological and economic advantages for recombinant HGH production.